Antiviral and Cytotoxic Agents from Ferula assa-foetida - American

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Influenza A (H1N1) Antiviral and Cytotoxic Agents from Ferula assa-foetida Chia-Lin Lee,† Lien-Chai Chiang,‡ Li-Hung Cheng,† Chih-Chuang Liaw,§ Mohamed H. Abd El-Razek,⊥ Fang-Rong Chang,*,† and Yang-Chang Wu*,† Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, Department of Microbiology, College of Medicine, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, Graduate Institute of Pharmaceutical Chemistry, China Medical UniVersity, Taichung 404, Taiwan, Republic of China, and Chemistry of Natural Products Department, National Research Center, Cairo, 12622, Egypt ReceiVed March 9, 2009

Two new sesquiterpene coumarins, designated 5′-acetoxy-8′-hydroxyumbelliprenin (1) and 10′R-acetoxy-11′-hydroxyumbelliprenin (2), and a new diterpene, 15-hydroxy-6-en-dehydroabietic acid (3), along with 27 known compounds, were isolated from a CHCl3-soluble extract of Ferula assa-foetida through bioassay-guided fractionation. The structures of the new metabolites 1-3 were identified by spectroscopic data interpretation and by the Mosher ester method. Compounds 4 and 6-13 showed greater potency against influenza A virus (H1N1) (IC50 0.26-0.86 µg/mL) than amantadine (IC50 0.92 µg/mL), and 11 exhibited the best potency (IC50 0.51, 2.6, and 3.4 µg/mL) of these compounds against the HepG2, Hep3B, and MCF-7 cancer cell lines, respectively. Influenza occurs with seasonal variations and reaches peak prevalence in winter, with many people killed worldwide every year. Until now, only a few organic compounds including amantadine, rimantadine, and ribavirin have been used for influenza therapy. However, drug-resistant influenza viruses are generated quickly. During the course of an anti-influenza and cytotoxicity screening program on natural products, it was found that a CHCl3 extract of Ferula assa-foetida was active against H1N1 influenza virus (IC50 < 3.4 µg/mL) and various human cancer cell lines (IC50 < 20 µg/mL). Therefore, the plant was selected as a lead to be investigated. F. assa-foetida L. (Apiaceae) is distributed mainly in Iran, Afghanistan, and mainland China. Nearly a hundred years ago, the roots of this plant were an important remedy for the well-known “Spanish Flu” (type A influenza, H1N1 subtype), which led to 20-100 million people dying at that time.1 The roots of F. assafoetida have been used for their perceived anthelmintic, anticarcinogenesis, anti-HIV, antimicrobial, antirheumatic, antispasmodic, diuretic, and emmenagogue actions in folk medicine.2-7 There are 236 natural products, including 129 sesquiterpene coumarins, seven monoterpene coumarins, 49 daucane sesquiterpenes, nine polysulfide derivatives, 10 sesquiterpene phenylpropanoids, 21 simple sesquiterpenes, six simple monoterpenes, and five oleanane triterpenes, reported from the genus Ferula to date. While F. assa-foetida has had extensive use in folk medicine for the treatment of influenza, it is interesting that the antiviral activities of the isolated compounds from this plant have not been evaluated in a systematic manner. Bioassay-directed chromatographic fractionation of F. assa-foetida resin led to the isolation of 30 compounds, including 20 sesquiterpene coumarins, 5′-acetoxy-8′-hydroxyumbelliprenin (1), 10′Racetoxy-11′-hydroxyumbelliprenin (2), 5′S-hydroxyumbelliprenin (4),8 10′R-karatavicinol (5),9 8′-acetoxy-5′S-hydroxyumbelliprenin (6),8,9 methyl galbanate (7),8,10,11 galbanic acid (8),10,11 farnesiferol C (9),10,12,13 farnesiferol A (10),12,13 farnesiferol B,12,13 assafoetidin,14 lehmferin,15 conferol (11),16 feselol,17 ligupersin A (12),18 epi-conferdione (13),9 microlobin,11 lehmannolone,19 polyanthinin,20 and kamolonol;21 two polysulfide derivatives, foetisulfide A and foetisulfide C;22 a simple coumarin, umbelliferone; a benzal* To whom correspondence should be addressed. Tel.: +886-7-312-1101, ext. 2197. Fax: +886-7-311-4773. E-mail: [email protected] or [email protected]. † Graduate Institute of Natural Products, Kaohsiung Medical University. ‡ Department of Microbiology, Kaohsiung Medical University. § China Medical University. ⊥ National Research Center, Cairo.

10.1021/np900158f CCC: $40.75

dehyde, vanillin; a drimane sesquiterpenoid, fetidone B;23 three diterpenes, 7-oxocallitrisic acid, picealactone C, and 15-hydroxy6-en-dehydroabietic acid (3); a sterol, β-sitosterol; and a fatty acid, oleic acid. Among them, two new sesquiterpene coumarins (1 and 2) and a new diterpene (3) were identified. Most of these compounds were evaluated in influenza A (H1N1) antiviral and cytotoxicity assays, and the results are described herein.

Results and Discussion A MeOH extract of the resin of F. assa-foetida was partitioned between n-hexane-MeOH (1:1), and then the MeOH layer was partitioned between CHCl3-H2O (1:1) to obtain a CHCl3 extract, which showed significant anti-H1N1 activity (IC50 < 3.4 µg/mL) and cytotoxicity for three cancer cell lines (IC50 < 20 µg/mL). Initial fractionation of the CHCl3 extract was carried out by open liquid chromatography on silica gel to give 11 fractions. Chromatographic fractionation of these active subfractions provided two new sesquiterpene coumarins (1 and 2), a new diterpene (3), and 27 known compounds. HRESIMS of 1 exhibited a [M + Na]+ ion at m/z 463.2099 (C26H32O6Na). The IR spectrum showed absorptions for hydroxy (3440 cm-1), acetoxy (1729 cm-1), and aromatic (1613 and 1555 cm-1) functional groups. UV absorptions at 211 and 318 nm also indicated a coumarin nucleus oxygenated at the C-7 position.12 In the 13C NMR spectrum, compound 1 displayed 26 carbon signals, with nine being typical for an umbelliferone skeleton [δ 101.5 (C8), 112.5 (C-10), 113.1 (C-3), 114.2 (C-6), 128.7 (C-5), 143.4 (C4), 155.8 (C-9), 161.2 (C-2), and 161.9 (C-7)] and the remaining 17 signals ascribable to a sesquiterpene moiety [δ 18.0 (C-13′), 13.1 (C-14′), 17.1 (C-15′), 25.9 (C-12′), 34.2 (C-9′), 44.9 (C-4′), 65.2 (C-1′), 68.9 (C-5′), 75.9 (C-8′), 119.5 (C-10′), 122.1 (C-2′), 123.3 (C-6′), 135.5 (C-11′), 137.6 (C-3′), 141.9 (C-7′)] with an acetoxy group (δ 21.2 and 170.3). In the 1H NMR spectrum, signals for two main moieties, a coumarin and a sesquiterpene, were revealed. The coumarin moiety appeared as five signals [δΗ 6.24 and 7.63 (each 1H, d, J ) 9.4 Hz), 7.35 (1H, d, J ) 8.4 Hz), 6.82 (1H, dd, J ) 2.4, 8.4 Hz), 6.79 (1H, d, J ) 2.4 Hz)]. In turn, the sesquiterpene moiety displayed signals for four methyls [δH 1.62, (3H, brs), 1.71 (3H, brs), 1.72 (3H, brs), 1.81, (3H, s)], two methylenes [δH 2.23 (2H, m), 2.28 (1H, d, J ) 6.0 Hz), 2.45 (1H, dd, J ) 7.6, 13.6 Hz)], an oxygenated methylene [δH 4.57 (2H, d, J ) 6.2 Hz)], three olefinic methines [δH 5.08 (1H, brt, J ) 6.4 Hz), 5.41 (1H, d, J ) 8.8 Hz), 5.50 (1H, brt, J ) 6.2 Hz)], and two oxygenated methines [δH 3.99 (1H, brt, J ) 6.4 Hz), 5.70 (1H,

 2009 American Chemical Society and American Society of Pharmacognosy Published on Web 08/19/2009

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Figure 1. Important HMBC correlations for compounds 1 and 2. Table 1. 1H and 13C NMR Data of Compounds 1 and 2 (400 and 100 MHz in CDCl3, δ in ppm, J in Hz) 1 position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ OAc

m)]. Compound 1 gave the same molecular formula, C26H32O6, and similar 1D NMR data to compound 6, 8′-acetoxy-5′-hydroxyumbelliprenin,12 with the only difference being due to the placement of an acetoxy group and a hydroxy group. The key HMBC correlations suggested that the acetoxy and hydroxy groups could be positioned at C-5′ and C-8′, respectively (Figure 1). The HMBC correlation of an oxygenated methylene at δH 4.57 (H-1′) with a carbon signal at δC 161.9 (C-7) indicated that the sesquiterpene unit is attached to C-7 of the coumarin moiety via an ether linkage. The absolute configuration at C-8′ was not determined due to the small amount of 1 available and also because no previous literature values could be used for comparison. The new compound 1 was therefore assigned as 5′-acetoxy-8′-hydroxyumbelliprenin. A [M + H]+ ion at m/z 443.2433 (C26H35O6) was present in the HRFABMS of 2. IR absorptions at 3480, 1731, and (1555 and 1613) cm-1 supported the presence of hydroxy, acetoxy, and aromatic ring functions, respectively. UV absorptions at 213 and 321 nm indicated a coumarin nucleus oxygenated at the C-7 position.12 In the 1D NMR spectrum (Table 1), the signals indicated a carbon skeleton with two main moieties, a coumarin and a sesquiterpene. On the basis of HMBC correlations [δH 4.59 (H-1′)/δC 162.1 (C7), 118.4 (C-2′), and 142.2 (C-3′); δH 4.77 (H-10′)/δC 171.2 (OCOCH3); δH 1.17 (H-12′)/δC 79.5 (C-10′), 72.4 (C-11′), and 26.7 (C-13′); δH 1.18 (H-13′)/δC 79.5 (C-10′), 72.4 (C-11′), and 24.8 (C-12′)], the sesquiterpene unit could be attached to C-7 (δ 162.1)

δH (J in Hz) 6.24 (d, 9.4) 7.63 (d, 9.4) 7.35 (d, 8.4) 6.82 (dd, 8.4, 2.4) 6.79 (d, 2.4)

4.57 (d, 6.2) 5.50 (brt, 6.2) 2.45 (brdd, 13.6, 7.6) 2.28 (d, 6.0) 5.70 (m) 5.41 (d, 8.8) 3.99 (brt, 6.4) 2.23 (m) 5.08 (brt, 6.4) 1.62 (brs) 1.71 (brs) 1.72 (brs) 1.81 (s) 1.99 (s) (C-5′)

2 δC 161.2 (s) 113.1 (d) 143.4 (d) 128.7 (d) 114.2 (d) 161.9 (s) 101.5 (d) 155.8 (s) 112.5 (s) 65.2 (t) 122.1 (d) 137.6 (s) 44.9 (t) 68.9 (d) 123.3 (d) 141.9 (s) 75.9 (d) 34.2 (t) 119.5 (d) 135.5 (s) 25.9 (q) 18.0 (q) 13.1 (q) 17.1 (q) 21.2 (q) 170.3 (s)

δH (J in Hz) 6.23 (d, 9.4) 7.63 (d, 9.4) 7.35 (d, 8.4), 6.82 (dd, 8.4, 2.4) 6.79 (d, 2.4)

4.59 (d, 6.4) 5.45 (brt, 6.4) 2.14-2.06 (2H, m) 2.14-2.06 (2H, m) 5.09 (brt, 6.2) 1.93 (2H, m) 1.68 (2H, m) 4.77 (dd, 10.0, 2.8) 1.17 (brs) 1.18 (brs) 1.58 (brs) 1.74 (brs) 2.10 (s) (C-10′)

δC 161.3 (s) 112.9 (d) 143.5 (d) 128.7 (d) 113.2 (d) 162.1 (s) 101.5 (d) 155.8 (s) 112.4 (s) 65.5 (t) 118.4 (d) 142.2 (s) 39.3 (t) 26.1 (t) 124.2 (d) 134.5 (s) 36.0 (t) 27.8 (t) 79.5 (d) 72.4 (s) 24.8 (q) 26.7 (q) 16.0 (q) 16.7 (q) 21.1 (q) 171.2 (s)

of the coumarin moiety via an ether linkage, and the acetoxy and hydroxy groups were positioned at C-10′ (δ 79.5) and C-11′ (δ 72.4), respectively. Compound 2 was elucidated as 10′-acetoxy11′-hydroxyumbelliprenin.24 To determine the only chiral center at C-10′, we speculated that 2 and 10′-karatavicinol (5) possess the same biogenetic origin. Consequently, 5 was treated separately with (R)- and (S)-Rmethoxy-R-(trifluoromethyl)phenylacetyl chloride [(R)- and (S)MTPA-Cl] in the presence of C5D5N, to yield the (S)- and (R)MTPA esters (5a and 5b), respectively.25 The MTPA esters were generated successfully at C-10′ as elucidated from the 1H NMR spectra (5a, H-8′ δ 2.15, H-9′ δ 2.15, H-10′ δ 5.49, H-12′ δ 1.37, H-13′ δ 1.70; 5b, H-8′ δ 2.13, H-9′ δ 2.13, H-10′ δ 5.50, H-12′ δ 1.43, H-13′ δ 1.71). The differences between the 1H NMR chemical shifts for 5a and 5b (∆ values shown in Figure 3) led to the assignment of the R-configuration at C-10′ of 5. From a comparison of the specific rotation data of 5 ([R]25D +16.2) and 2 ([R]25D +11.2), the absolute configuration of 2 at C-10′ could therefore be assigned with an R-configuration. Compound 2 was established as 10′R-acetoxy-11′-hydroxyumbelliprenin. Compound 3 gave the molecular formula C20H26O3, as determined by HRESIMS (m/z 337.1784 [M + Na]+), indicating eight degrees of unsaturation. Its IR spectrum showed absorptions attributable to hydroxy (3417 cm-1), carboxylic acid (1696 cm-1), and aromatic ring (1613 and 1514 cm-1) functions. On the basis of its 13C NMR and DEPT data, 3 showed 20 carbon signals,

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Lee et al. Table 3. Influenza A (H1N1) Antiviral Activity for Compounds 2 and 4-13

Figure 2. Important HMBC correlations for compound 3.

Figure 3. 1H NMR chemical shift differences [∆δ ) δ H(S)-MTPA - δ H(R)-MTPA (ppm)] of the MTPA esters of 5 and 6. Table 2. 1H and 13C NMR Data of Compound 3 (400 and 100 MHz in CDCl3, δ in ppm, J in Hz) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

δH (J in Hz) 2.21 (brd, 11.2) 1.81 (m) 1.81 (m) 1.76 (m) 2.91 (t, 2.8) 5.81 (dd, 9.6, 2.7) 6.55 (dd, 9.6, 2.9)

7.13 (d, 8.4) 7.30 (dd, 8.4, 2.0) 7.17 (d, 2.0) 1.57 (brs) 1.57 (brs) 1.40 (s) 1.08 (s)

δC 35.2 (t) 18.3 (t) 35.6 (t) 46.0 (s) 46.3 (d) 130.0 (d) 128.3 (d) 132.4 (s) 145.9 (s) 37.1 (s) 121.6 (d) 123.7 (d) 146.6 (s) 122.8 (d) 72.4 (s) 31.6 (q) 31.6 (q) 183.5 (s) 17.7 (q) 20.7 (q)

including four methyls (δC 17.7, 20.7, 31.6, 31.6), three methylenes (δC 18.3, 35.2, 35.6), six methines (δC 46.3, 121.6, 122.8, 123.7, 128.3, 130.0), and seven quaternary (δC 37.1, 46.0, 72.4, 132.4, 145.9, 146.6, 183.5) carbons (Table 2). Among the seven quaternary carbons, one was assigned as a carbonyl carbon at δ 183.5. Therefore, the data supported the presence of one carbonyl, four olefins, and three ring moieties to fulfill the eight degrees of apparent unsaturation, and 3 was postulated to be an abietane-type diterpene.26 The HMBC correlations [δH 1.57 (H-16)/δC 72.4 (C-15), 31.6 (C-17), and 146.6 (C-13); δH 1.57 (H-17)/δC 72.4 (C-15), 31.6 (C-16), and 146.6 (C-13); δH 1.40 (H-19)/δC 35.6 (C-3), 46.0 (C4), 46.3 (C-5), and 183.5 (C-18)] suggested the hydroxy and carboxylic groups to be located at C-15 (δ 72.4) and C-4 (δ 46.0), respectively (Figure 2). The NOESY correlation between Me-19 and Me-20 as well as the absence of NOESY correlations between

compound

IC50 (µg/mL)

IC90 (µg/mL)

2 4 5 6 7 8 9 10 11 12 13 amantadine

0.94 ( 0.07 0.36 ( 0.03 0.99 ( 0.07 0.81 ( 0.06 0.26 ( 0.03 0.45 ( 0.04 0.29 ( 0.02 0.51 ( 0.04 0.47 ( 0.05 0.86 ( 0.08 0.32 ( 0.02 0.92 ( 0.04

1.98 ( 0.12 0.62 ( 0.08 2.05 ( 0.14 1.56 ( 0.11 0.44 ( 0.05 0.88 ( 0.07 0.50 ( 0.07 0.64 ( 0.07 0.83 ( 0.09 1.75 ( 0.12 0.37 ( 0.03 1.73 ( 0.11

H-5/Me-19 and H-5/Me-20 indicated β- and R-orientations, respectively, for Me-20 and H-5 (trans A/B ring junction). Compound 3 was therefore elucidated as 15-hydroxy-6-en-dehydroabietic acid. To the best of our knowledge, this is the first report of a diterpene with an abietane skeleton from the genus Ferula. On the basis of the work of Nittala et al.,26 the chiral center of the known 5′-hydroxyumbelliprenin (4) is similar to that of 12,13didehydrofurospongin-1. The absolute configuration, the S form, of 12,13-didehydrofurospongin-1 was determined by the Mosher method. From a comparison of their specific rotation data {12,13didehydrofurospongin-1 ([R]28D -8); 4 ([R]25D -3.8)}, the absolute configuration of 4 at C-5′ was assigned the S form. Compound 4 is accordingly 5′S- hydroxyumbelliprenin. The absolute configuration of the known 8′-acetoxy-5′-hydroxyumbelliprenin (6)12 at C-5′ was still unknown at the time of the present study. Compound 6 was treated separately with (R)- and (S)-MTPA-Cl in C5D5N to yield the (S)- and (R)-MTPA esters (6a and 6b), respectively. The MTPA esters were generated successfully at C-5′, as elucidated from the 1H NMR spectra (6a, H-2′ δ 5.58, H-4′a δ 2.37, H-4′b δ 2.31, H-5′ δ 6.20, H-6′ δ 5.70, H-8′ δ 5.35, H-14′ δ 1.93, H-15′ δ 1.72; 6b, H-2′ δ 5.69, H-4′a δ 2.41, H-4′b δ 2.35, H-5′ δ 6.13, H-6′ δ 5.49, H-8′ δ 5.30, H-14′ δ 1.79, H-15′ δ 1.94). The differences between the 1H NMR chemical shifts for 6a and 6b (∆ values shown in Figure 3) led to the assignment of the S-configuration at C-5′, and 6 was determined as 8′-acetoxy5′S-hydroxyumbelliprenin. Sesquiterpene coumarin ethers (7-hydroxycoumarin analogues) have been reported as possessing various biological activies, including potent NF-κB inhibition [8′-acetoxy-5′S-hydroxyumbelliprenin (6)],22 antibacterial activity [galbanic acid (8)],27 a potential cancer chemopreventive effect [farnesiferol C (9)],28 squalenehopene cyclase inhibition [karatavicinol (5) and farnesiferol C (9)],29 and human rhinovirus coat protein inhibition [karatavicinol (5) and farnesiferol B].30 Since 7-hydroxycoumarin analogues showed good anti-HIV activity in previous studies,3,22 the pure 7-O-sesquiterpene coumarins, compounds 2 and 4-6 (acyclic), 7-9 (monocyclic), and 10-13 (bicyclic), were screened in an in vitro anti-influenza A viral (H1N1) assay, with amantadine as a positive control (Table 3). Most of these compounds exhibited a higher antiviral potency than amantadine, except for 2, 5, and 12. These results are of interest because the structures of many of these compounds are quite similar. For example, 6 (OH-5′, OAc-8′) has an additional acetoxy group in comparison with 4 (OH-5′), but 6 was much less potent than 4. Compound 2 (OAc-10′) has the same skeleton as 5 (OH-10′), and these two compounds showed a similar potency to the positive control, amantadine. Between compounds 7 (COOCH3-3′) and 8 (COOH-3′), 7 showed better potency than 8 and, therefore, indicated that methyl esterification of C-3′ enhanced the activity in the bioassay used. For the bicyclic-sesquiterpene coumarins, 12 and 13, a C-3′-carbonyl afforded greater potency than a C-3′-OH in this kind of skeleton. Overall, the present study has determined that sesquiterpene coumarins from F. assa-foetida may serve as

AntiViral and Cytotoxic Agents from Ferula assa-foetida promising lead compounds for new drug development against influenza A (H1N1) viral infection. A standardized plant extract of F. assa-foetida, a species that has been used to treat influenza for many years, may also be worthy of being further investigated as a new phytomedicine. Furthermore, the pure compounds were screened in a cytotoxicity assay with doxorubicin as the positive control. The minor compound 11 exhibited the best potency (IC50 0.51, 2.6, and 3.4 µg/mL) against HepG2, Hep3B, and MCF-7 tested cancer cell lines, respectively. Interestingly, compound 11 also showed high potency influenza activity (IC50 0.47 ( 0.05 µg/mL). The remaining compounds were all inactive for all cancer cell lines (IC50 > 4 µg/mL; Table S1, Supporting Information). Experimental Section General Experimental Procedures. Optical rotations were taken on a JASCO-P-1020 polarimeter (cell length 10 mm). UV spectra were measured on a JASCO V-530 UV/vis spectrophotometer. IR spectra were recorded on a Mattson Genesis II FT-IR spectrophotometer. NMR spectra were recorded on Varian Gemini-20000 (200 MHz), Varian Unity-plus (400 MHz), and Varian Unity-plus (600 MHz) FT-NMR NMR spectrometers. Chemical shift (δ) values are in ppm (parts per million), with CDCl3 as internal standard, and coupling constants (J) are in Hz. HRFABMS, HRESIMS, and ESIMS measurements were performed on JEOL JMS-700, Bruker APEX II, and Finnigan POLARISQ mass spectrometers. TLC was performed on Kieselgel 60, F254 (0.20 mm, Merck), and spots were viewed under UV light at 254 and 356 nm and/or stained by spraying with 50% H2SO4 and heating on a hot plate. For column chromatography, silica gel (Kieselgel 60, 70-230 and 230-400 mesh, Merck) and Sephadex LH-20 were used. The instrumentation for the RP-MPLC experiment was composed of a Supelco VersaFlash flash chromatography apparatus and VersaFlash C-18 cartridges (40 × 150 mm). Further purification of some compounds obtained was achieved by preparative HPLC, using a Shimadzu LC-10ATVp/Shimadzu SCL-10AVp UV-vis detector, and Thermo columns (analytical: 5 µm, 250 × 4.6 mm; preparative: 8 µm, 250 × 10 mm; C18) were used. For the preparation of Mosher ester derivatives, (S)-(+)- and (R)-(-)-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride were used as the reagents. Plant Material. Ferula assa-foetida resin (3.23 kg) was purchased from a Chinese herb shop in Taipei, Taiwan, in November 2006 and identified by one of the authors (M.H.A.R.). A voucher specimen (FA200611) was deposited at the Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan. Extraction and Isolation. F. assa-foetida resin (3.23 kg) was extracted four times with MeOH (5 L each) at room temperature to obtain a crude extract (313.8 g). The crude extract was partitioned between n-hexane-MeOH (1:1), and then the MeOH extract (120.1 g) was partitioned between CHCl3-H2O (1:1), to obtain a CHCl3 extract (65.2 g), which showed significant anti-H1N1 antiviral and cytotoxic activities. Initial fractionation of the CHCl3 extract (65.2 g) was carried out by open column chromatography on silica gel (230-400 mesh, column: 7 × 30 cm), using gradients of n-hexane-CHCl3-MeOH (100: 0:0 to 0:80:20), and gave 11 fractions. A precipitation was obtained from the first chromatographic step, using n-hexane as the eluent, and was washed with a trace amount of n-hexane to afford oleic acid (2.0 g). Fraction 3 (6.66 g) was fractionated into 15 fractions by silica gel chromatography (70-230 mesh, column: 5 × 23 cm; CHCl3). Subfraction 3-6 (602.06 mg) was subjected to silica chromatography (70-230 mesh, column: 3 × 20 cm; n-hexane-EtOAc, 1:1) and purified by preparative TLC (CHCl3) to give 9 (1.9 mg). Subfraction 3-7 (619.90 mg) was purified by solid-phase extraction (SPE) (DSC18, 60 mL, 10GMS, No. 52609-U; MeOH-H2O, 80:20) to obtain vanillin (23.5 mg). Fraction 4 (11.78 g) was separated into eight subfractions by column chromatography on silica gel (70-230 mesh, column: 7 × 24 cm; n-hexane-EtOAc, 1:1), and crystalline β-sitosterol (0.30 g) was obtained with MeOH. Subfraction 4-6 was subjected to silica gel chromatography (70-230 mesh, column: 5 × 17 cm; CHCl3-MeOH, 60:1) to give six subfrations. Subfraction 4-6-2 was purified with HPLC (ODS Thermo, 10 × 250 mm; MeOH-H2O, 78:22; flow rate: 3.0 mL/ min) to give lehmannolone (1.7 mg, tR ) 31 min). Subfraction 4-6-4

Journal of Natural Products, 2009, Vol. 72, No. 9 1571 was purified by preparative TLC (CHCl3) to give 4 (5.7 mg) and lehmferin (2.8 mg). Subfraction 4-6-5 (400 mg) was chromatographed on a silica gel column (70-230 mesh, column: 2.5 × 28 cm), using n-hexane-CH2Cl2 (2:7) as eluent, and subfraction 4-6-5-1 (253.30 mg) was purified subsequently with n-hexane-EtOAc (7:2), to obtain seven subfractions. Subfractions 4-6-5-1-2 (9.5 mg), 4-6-5-1-3 (68.7 mg), and 4-6-5-1-6 (21.9 mg) were purified by RP-HPLC (flow rate: 3.0 mL/ min) to give fetidone B (2.2 mg, MeOH-H2O, 70:30, tR ) 14 min), a farnesiferol B and assafoetidin mixture (16.4 mg, MeOH-H2O, 63: 37, tR ) 123 min), feselol (3.3 mg, MeOH-H2O, 63:37, tR ) 136 min), and 1 (2.3 mg, MeOH-H2O 70:30, tR ) 34 min), respectively. Subfractions 4-6-5-2 (43.4 mg) and 4-6-7 (354.7 mg) were purified by RP-HPLC (MeOH-H2O, 76:24; flow rate: 3.0 mL/min) to give 10 (8.9 mg, tR ) 38 min) and 11 (6.2 mg, tR ) 41 min), and 7 (58.9 mg, tR ) 27 min), 8 (15.7 mg, tR ) 31 min), and 7-oxocallitrisic acid (1.5 mg, tR ) 53 min), respectively. Subfraction 4-7 (12.40 g) was separated by silica gel chromatography (70-230 mesh, column: 5 × 25 cm; CHCl3-MeOH, 60:1) into six subfractions. Subfraction 4-7-4 (746.80 mg) was subjected to silica gel chromatography (70-230 mesh, column: 3 × 20 cm; n-hexane-EtOAc, 5:2) and purified by RP-HPLC (MeOH-H2O, 75:25; flow rate: 3.0 mL/min) to give 2 (19.7 mg, tR ) 24 min), 6 (57.0 mg, tR ) 34 min), and 13 (11.6 mg, tR ) 12 min). The CHCl3/MeOH-insoluble umbelliferone (3.4 mg) was obtained from subfraction 4-8-7 (58.2 mg). Fraction 5 (11.77 g) was chromatographed over silica gel (70-230 mesh, column: 5 × 30 cm; CHCl3-MeOH, 50:1), with subfraction 5-2 (334.30 mg) then subjected to RP-MPLC (MeOH-H2O, 65:35), to give five subfractions. Subfraction 5-2-1 was purified by RP-HPLC (MeOH-H2O, 61:39; flow rate: 3.0 mL/min) to give foetisulfide A (1.5 mg, tR ) 43 min). Subfraction 5-3 (255.2 mg) was subjected to RP-MPLC (MeOH-H2O, 70:30) and subfraction 5-3-3 (112.3 mg) was purified by silica gel chromatography (70-230 mesh, column: 2.5 × 30 cm; CH2Cl2-MeOH, 40:1) to obtain kamolonol (14.3 mg). Subfraction 5-3-4 (351.4 mg) was chromatographed by RP-HPLC (MeOH-H2O, 65:35; flow rate: 3.0 mL/min) to give microlobin (30.4 mg, tR ) 73 min), and then the mother liquor was purified by RPHPLC (MeOH-H2O, 53:47; flow rate: 3.0 mL/min) to give 12 (21.3 mg, tR ) 64 min) and foetisulfide C (3.6 mg, tR ) 50 min). Subfractions 5-3-5 and 5-3-6 were chromatographed over silica gel (70-230 mesh, column: 1.5 × 25 cm; CH2Cl2-MeOH, 60:1 and 50:1) to give picealactone C (0.7 mg) and 5 (241.7 mg). Subfraction 5-5 (352.3 mg) was chromatographed by RP-HPLC (MeOH-H2O, 72:28; flow rate: 3.0 mL/min) to give 3 (7.0 mg, tR ) 16 min). Fraction 6 (4.33 g) was chromatographed over silica gel (70-230 mesh, column: 7 × 35 cm; CH2Cl2-MeOH, 25:1) to give five subfractions. The third subfraction was purified with RP-MPLC (MeOH-H2O, 55:45) to give polyanthinin (23.4 mg). 5′-Acetoxy-8′-hydroxyumbelliprenin (1): yellow oil; [R]25D +8.6 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 211 (4.54), 318 (3.95) nm; IR (neat) νmax 3440, 1729, 1613, 1555, 1233, 836 cm-1; 1H and 13C NMR (CDCl3, 400 MHz), see Table 1; ESIMS m/z 463 [M + Na]+; HRESIMS m/z 463.2099 [M + Na]+(calcd for C26H32O6Na, 463.2096). 10′R-Acetoxy-11′-hydroxyumbelliprenin (2): yellow oil; [R]25D +11.2 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 213 (4.77), 321 (4.39) nm; IR (neat) νmax 3480, 1731, 1613, 1555, 1234, 836 cm-1; 1H and 13 C NMR (CDCl3, 400 MHz), see Table 1; ESIMS m/z 465 [M + Na]+; HRFABMS m/z 443.2433 [M + H]+ (calcd for C26H34O6 + H, 443.2434). 15-Hydroxy-6-en-dehydroabietic acid (3): yellow oil; [R]25D -14.6 (c 0.16, CHCl3); UV (MeOH) λmax (log ε) 243 (3.95), 271 (3.74) nm; IR (neat) νmax 3417, 1696, 1613, 1514 cm-1; 1H and 13C NMR (CDCl3, 400 MHz), see Table 2; ESIMS m/z 337 [M + Na]+; HRESIMS m/z 337.1784 [M + Na]+ (calcd for C20H26O3Na, 337.1780). Anti-influenza A Virus (H1N1) Bioassay.31,32 Madin-Darby canine kidney (MDCK) cells (ATCC CCL34) were used as target cells for viral infection in the XTT (tetrazolium hydroxide salt) assay. They were grown as adherent cells in MEM medium supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin G, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. In the antiviral assay, the medium was supplemented with 2% FCS and the above-mentioned antibiotics. Virus titers were determined by the cytopathic effect in MDCK cells and expressed as 50% tissue culture infective dose (TCID50) values per mL. All viruses were stored at -70 °C until use. The antiviral activity against influenza A virus (H1N1) was evaluated by the XTT method. MDCK cells, treated by trypsin, were seeded onto 96-well

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plates with a concentration of 1.0 × 105 cells per mL and a volume of 70 µL per well. After incubation at 35 °C with 5% CO2 for 24 h, 20 µL of test virus solution was added and incubated for another 1 h. Different concentrations of test substances were then added to culture wells in triplicate. Amantadine was used as a positive control. After incubation at 35 °C with 5% CO2 for 3 days, XTT reagent was added and incubated for 3 h. The viral inhibition rate (%) was calculated as [100 - (OD492/OD690) × 100]%. The antiviral concentration of 50% inhibition (IC50) was defined as the concentration achieving 50% cytoprotection against virus infection. Cytotoxicity Bioassays. Fractions and isolates were tested against lung (A549), breast (MEA-MB-231 and MCF7), liver (HepG2 and Hep3B), and oral (Ca9-22) human cancer cell lines using an established colorimetric MTT (diphenyltetrazolium bromide) assay protocol.33 Doxorubicin was used as a positive control. In brief, freshly trypsinized cell suspensions were seeded in 96-well microtiter plates at densities of 5000-10 000 cells per well with test compounds added from a DMSO stock solution. After 3 days in culture, the attached cells were incubated with MTT (0.5 mg/mL, 1 h) and subsequently solubilized in DMSO. The absorbance was measured at 550 nm using an ELISA reader. The IC50 is the concentration of agent that reduced cell growth by 50%, under the experimental conditions used. Acknowledgment. This work was supported by grants from the National Science Council and the Committee on Chinese Medicine and Pharmacy, Department of Health, Executive Yuan, Taiwan, awarded to Y.-C.W. and F.-R.C. Supporting Information Available: 1H, 13C, HSQC, HMBC, COSY, and NOESY spectra of compounds 1-3 along with cytotoxicity data. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Krebs, T. E. Bulletin of the NeVada State Board of Health; Carson City, NV, 1920; No. 1, pp 7-9. (2) Zhou, P.; Takaishi, Y.; Duan, H.; Chen, B.; Honda, G.; Itoh, M.; Takeda, Y.; Kodzhimatov, O. K.; Lee, K. Phytochemistry 2000, 53, 689–697. (3) Lee, T. T. Y.; Kashiwada, Y.; Huang, L.; Snider, J.; Cosentino, M.; Lee, K. H. Bioorg. Med. Chem. 1994, 2, 1051–1056. (4) Appendino, G.; Cravotto, G.; Sterner, O.; Ballero, M. J. Nat. Prod. 2001, 64, 393–395. (5) Iranshahi, M.; Kalategi, F.; Rezaee, R.; Shahverdi, A. R.; Ito, C.; Furukawa, H.; Tokuda, H.; Itoigawa, M. Planta Med. 2008, 74, 147– 150. (6) Eigner, D.; Scholz, D. J. Ethnopharmacol. 1999, 67, 1–6. (7) Banerji, A.; Malliok, B.; Chatterjee, A. Tetrahedron Lett. 1988, 29, 1557–1560. (8) Appendino, G.; Tagliapietra, S.; Nano, G. M.; Jakupovic, J. Phytochemistry 1994, 35, 183–186.

Lee et al. (9) Nabiev, A. A.; Khasanov, T. K.; Malikov, V. M. Khim. Prir. Soedin. 1982, 578–581; Chem. Abstr. 1983, 98, 86223. (10) Yang, J. R.; An, Z.; Jing, S.; Qin, H. L. Chem. Pharm. Bull. 2006, 54, 1595–1598. (11) Bizhanova, K.; Saidkhodzhaev, A. I. Khim. Prir. Soedin. 1978, 265– 267; Chem. Abstr. 1978, 89, 56443. (12) Kuliev, Z. A.; Khasanov, T. K.; Malikov, V. M. Khim. Prir. Soedin. 1982, 120–121; Chem. Abstr. 1982, 96, 177967. (13) Abd E1-Razek, M. H.; Wu, Y. C.; Chang, F. R. C. J. Chin. Chem. Soc. 2007, 54, 235–238. (14) Caglioti, L.; Naef, H.; Arigoni, D.; Jeper, O. HelV. Chem. Acta 1959, 42, 2557–2570. (15) Sagitdinova, G. V.; Saidkhodzhaev, A. I.; Malikov, V. M. Khim. Prir. Soedin. 1983, 709–712; Chem. Abstr. 1984, 100, 171545. (16) Bagirov, V. Yu.; Gasanova, R. Yu.; Burma, O. I.; Ban’kovskii, A. I. Khim. Prir. Soedin. 1977, 279–280; Chem. Abstr. 1977, 87, 81275. (17) Nabiev, A. A.; Khasanov, T. Kh.; Melibaev, S. Khim. Prir. Soedin. 1978, 517–518; Chem. Abstr. 1978, 89, 211931. (18) Nabiev, A. A.; Malikov, V. M. Khim. Prir. Soedin. 1983, 700–704; Chem. Abstr. 1984, 100, 171543. (19) Appendino, G.; Maxia, L.; Bascope, M.; Houghton, P. J.; SanchezDuffhues, G.; Munoz, E.; Sterner, O. J. Nat. Prod 2006, 69, 1101– 1104. (20) Vandyshev, V. V.; Sklyar, Yu. E.; Perel’son, M. E.; Moroz, M. D.; Pimenov, M. G. Khim. Prir. Soedin. 1972, 670–671; Chem. Abstr. 1973, 78, 108265. (21) Duan, H.; Takaishi, Y.; Tori, M.; Takaoka, S.; Honda, G.; Ito, M.; Takeda, Y.; Kodzhimatov, O. K.; Kodzhimatov, K.; Ashurmetov, O. J. Nat. Prod. 2002, 65, 1667–1669. (22) Banerji, A.; Mallick, B.; Chatterjee, A.; Budzikiewicz, H.; Breuer, M. Tetrahedron Lett. 1988, 29, 1557–1560. (23) Caglioti, L.; Naef, H.; Arigoni, D.; Jeper, O. HelV. Chem. Acta 1958, 41, 2278–2292. (24) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092–4096. (25) Kobayashi, M.; Chavakula, R.; Murata, O.; Sarma, N. S. Chem. Pharm. Bull. 1992, 40, 599–601. (26) Gigante, B.; Silva, A. M.; Marcelo-Curto, M. J.; Feio, S. S.; Roseiro, J.; Reis, L. V. Planta Med. 2002, 68, 680–684. (27) Shahverdi, A. R.; Fakhimi, A.; Zarrini, G.; Dehghan, G.; Iranshahi, M. Biol. Pharm. Bull. 2007, 30, 1805–1807. (28) Pillai, S. P.; Menon, S. R.; Mitscher, L. A.; Pillai, C. A.; Shankel, D. M. J. Nat. Prod. 1999, 62, 1358–1362. (29) Gravotto, G.; Balliano, G.; Robaldo, B.; Oliaro-Bosso, S.; Chimichi, S.; Boccalini, M. Bioorg. Med. Chem. Lett. 2004, 14, 1931–1934. (30) Rollinger, J. M.; Steindl, T. M.; Schuster, D.; Kirchmair, J.; Anrain, K.; Ellmerer, E. P.; Langer, T.; Stuppner, H.; Schmidtke, M. J. Med. Chem. 2008, 51, 842–851. (31) Chiang, L. C.; Chiang, W.; Chang, M. Y.; Ng, L. T.; Lin, C. C. AntiViral Res. 2002, 55, 53–62. (32) Kodama, E.; Shigeta, S.; Suzuki, T.; De Clercq, E. AntiViral Res. 1996, 31, 159–164. (33) Chan, M. L.; Chen, W. Y.; Tsai, C. Y.; Chang, F. R.; Wu, Y. C. Mol. Cancer Ther. 2005, 4, 1277–1285.

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